This invention is directed to a process for the isolation of components such as nucleic acids from natural sources by removing the digested natural sources such as cells or cell debris in a sample by filtration, and to a device for operating said process.
Frequently, in the preparation of cell components, particularly nucleic acids, the problem arises to separate the digested natural sources, from which the components are derived, from dissolved material. Removal of cells or cell debris is effected by centrifugation, whereby larger cell fragments or cells deposit as a pellet in the centrifugation tube. The cell components then are found in the supernatant and may be pipetted. Filtration procedures which are simpler per se were not capable of prevailing because the digested cells or fragments thereof either pass through the filter having too large a pore size and thus, give rise to turbidity and impurities in the filtrate or, when using filters with appropriately narrow pores, however, inevitable jamming results, so that purposeful preparation of the cell components is no longer possible.
Thus, the present invention is based on the problem of providing a process and creating a device by means of which centrifugation steps for the preparation of cell components from natural sources such as cells may be avoided by using filtration steps which are easier to handle.
The technical problem which the invention is based upon is solved by a process according to the features of claim 1. The subsequent subclaims are directed to preferred embodiments of the process according to the invention. The device of the invention presents the features of claim 10. The subsequent subclaims are directed to preferred embodiments of the device according to the invention.
Conventionally, in order to isolate the components from cells, the latter are digested first. In the preparation of nucleic acids, the cells have to be digested first by using enzymes such as, for instance, proteinase K and lysozyme, detergents such as SDS, Brij, Triton-X-100, Tween 20, and DOC, and chemicals such as sodium hydroxide, guanidine hydrochloride and guanidine isothiocyanate. Thereafter, the thus processed sample material is subjected to filtration, wherein the pore size of the filter used in filtration decreases in the direction of sample flow.
In a preferred embodiment, sample flow during filtration may be facilitated by applying elevated pressure or reduced pressure. However, due to the pore size configuration of the filter, passage of the sample to be filtrated through the filter is also possible solely by gravity as the driving force. Furthermore, in order to make the passage of sample through the filter more rapid, the sample may also be passed through the filter by centrifugation.
In particular, the process according to the invention is suitable for the preparation of plasmid DNA or genomic DNA having a size of from 1 to 50 kb.
As the filters which may be used in the process according to the invention, there are possible, in particular, those made of sintered polyethylene, polypropylene, polytetrafluoroethylene, glass, silica gel, aluminum oxide or packed diatomaceous earth, e.g., Cellit or silica gel, woven or bonded fleeces of polypropylene, polyester, glass fiber, and silica, as well as paper, pressed paper, fleeces made of paper or combinations thereof.
In a preferred embodiment of the process according to the invention, multiple samples are processed simultaneously and passed through appropriate devices advantageously adapted to microtitration systems.